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SN Applied Sciences (2019) 1:355 | https://doi.org/10.1007/s42452-019-0375-x

Research Article

Enhancement ofantibacterial andanticancer properties ofpure andREM doped ZnO nanoparticles synthesized using Gymnema sylvestre leaves extract

M.Karthikeyan1 A.JafarAhamed1 C.Karthikeyan2 P.VijayaKumar3

Springer Nature Switzerland AG 2019

AbstractZinc oxide (ZnO) (K1) doped with rare earth metals (REM) such as lanthanum doped ZnO (K2), cerium doped ZnO (K3) and neodymium doped ZnO (K4) nanoparticles (NPs) were synthesized by green method using Gymnema sylvestre (G. sylvestre) leaves extract as reducing as well as capping agent and this method was also one of the alternatives to conven-tional physical and chemical methods. The synthesized K1, K2, K3 and K4 samples were characterized by X-ray diffraction analysis (XRD), X-ray photoelectron spectroscopy (XPS), field emission scanning electron microscopy (FESEM), energy dispersive X-ray spectroscopy (EDAX), Fourier transform infrared spectroscopy (FTIR), UVvisible spectroscopy, photo-luminescence (PL) techniques and electron para magnetic resonance (EPR) spectroscopy. The K1, K2, K3 and K4 samples were tested against clinical pathogens such as gram positive G+ (Staphylococcus aureus and Streptococcus pneumoniae) and gram negative G (Klebsiella pneumoniae, Shigella dysenteriae, Escherichia coli, Pseudomonas aeruginosa and Proteus vulgaris) bacterial strains using well diffusion method. The K2 sample shows higher antibacterial effect when compared to K1, K3 and K4 samples. Invitro cytotoxicity effect was analysed for A498 (human kidney carcinoma) cell line and normal vero (African monkey kidney) cell lines.

Keywords ZnO nanoparticles Gymnema sylvestre Rare earth metals XPS EPR Antibacterial and anticancer

1 Introduction

Recently, rare earth metal (REM) ions doped semiconduc-tor NPs are used in various fields such as optical, electronic and magnetic devices [16]. Since, ZnO NPs exhibit unique characteristics like low cost, nontoxicity, eco-friendly sys-tem to the nature and easy to prepare compounds with various morphologies having different properties. How-ever, ZnO NPs is an n-type semiconductor with wide direct band gap (3.37eV) and large excitation binding energy (60meV) at room temperature.

Nano sized ZnO can be potentially important with numerous applications such as solar cells [7], gas sensors [8], photocatalytic, antibacterial, electrical and optical

devices [9], electrostatic dissipative coatings [10], deg-radation of environmental pollutants [11, 12] and exter-nal uses as antibacterial agents in lotions, mouthwashes, ointments and surface coatings to prevent microbial growth [13]. The REM-doped ZnO NPs are vibrant mate-rials for flat panel displays for efficient emission in the visible range. On other hand, ZnO NPs is one of the envi-ronmental friendly materials. The REM-doped ZnO NPs may be used as florescence labels for biological medical imaging [14].

Chemically synthesized nanoparticles by-products are toxic to the environment [15]. Among this, to avoid the toxic by-products green synthesis using the bio-materi-als such as microorganisms and plants or plant extracts

Received: 9 January 2019 / Accepted: 18 March 2019 / Published online: 22 March 2019

* A. Jafar Ahamed, [email protected] | 1Department ofChemistry, Jamal Mohamed College (Autonomous), Tiruchirappalli, TamilNadu620020, India. 2KIRND Institute ofResearch andDevelopment Pvt Ltd., Tiruchirappalli, TamilNadu620020, India. 3Department ofChemistry, Asan College ofArts andScience, Karur, TamilNadu639003, India.
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Research Article SN Applied Sciences (2019) 1:355 | https://doi.org/10.1007/s42452-019-0375-x

derived metal oxide nanoparticles can be an effective alternative method for chemical synthesis. Various bio-logical sources are used for green synthesis like plant [16], bacteria [17], fungi [18] and yeast [19].

Among them G. sylvestre belongs to Asclepiadaceae family group, which is potentially used for treatment of asthma, eye complaints and snake bites [20]. This leaves extract holds a large number of bioactive compounds such as benzene-1,2-diol, 3-Allyl-2-methoxyphenol, hexadecanoic acid, octa decanoic acid [21], saponins (Gymnemic acid) and tannins [22]. Vijaya Kumar etal. have reported that Gymnemic acid has been a good reducing agent to prepare the metal oxide nanoparti-cles [23].

The metal oxide nanomaterials have been potentially used in biomedical applications, which may be due to the high surface area of metal oxide NPs. It has consid-erably enhanced its ability to produce reactive oxygen species (ROS) [24, 25]. ROS production may be effected by various paths such as irradiance of nanomaterials by ultraviolet (UV) light, disturbance of intracellular meta-bolic activities, and antioxidant system. This results in the generation of oxidative stress in the cells. ROS can cause cell death in the DNA, cell membrane, and pro-teins [26, 27].

In the present investigation, pure and REM ions (La3+, Ce3+, and Nd3+) doped ZnO NPs were synthesized by green method using G. sylvestre leaves extract. The

structural, optical, antibacterial and anticancer properties of the pure and REM-doped ZnO NPs have been studied in this work.

2 Experimental methods

2.1 Synthesis ofpure andREM doped ZnO NPs byusing G. sylvestre leaves extracts

Gymnema sylvestre leaves was taken and washed several time with tap and double distilled water. After that 15g of leaves was taken in 150ml of deionized water in a beaker and boiled at 80C for 1h. The prepared leaves extract was filtered using Whatman-1 filter paper.

In the case of ZnO NPs, 0.1M Zn(NO3)26H2O solution was dissolved into 150ml of G. sylvestre leaves extract. Homogenously mixed nitrate solution was continuously stirred at 80C for 6h. An yellow colour precipitate was obtained. Further the precipitate was dried at 120C for 2h. The obtained ZnO nanopowder were annealed at 700C for 5h and stored in an airtight container.

Similarly, for La-doped ZnO sample, 0.002 M La(NO3)36H2O was added into 0.098 M Zn(NO3)26H2O and dissolved in 150ml of G. sylvestre leaves extract and the above homogeneously mixed solution was stirred constantly at 80C for 6h. The yellow colour precipitate obtained, was dried at 120C for 2h, to get La doped ZnO

Fig. 1 Schematic diagram for the formation of K1, K2, K3 and K4 samples

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SN Applied Sciences (2019) 1:355 | https://doi.org/10.1007/s42452-019-0375-x Research Article

2.2 Antibacterial assay

The antibacterial activity of the K1, K2, K3 and K4 sam-ples was studied against gram positive G+ (Staphylo-coccus aureus and Streptococcus pneumoniae) and gram negative G (Klebsiella pneumoniae, Shigella dysenteriae, Escherichia coli, Pseudomonas aeruginosa and Proteus vul-garis) bacterial strains using well diffusion method. Petri plates were prepared with 25ml of sterile Muller Hin-ton agar (MHA, Himedia) and each bacterial pathogen was individually swabbed on MHA in separate plates. The antibacterial activity was tested at a concentra-tion of 1.5mg/ml with the required quantity of the NPs dispersed in dimethyl sulphoxide (DMSO). The zone of inhibition levels (mm) were measured after 24h and before this step, it was incubated overnight at 37C. The standard antibiotic Amoxicillin was used as the positive control.

2.3 Cell culture

A498 (kidney carcinoma cell) and Vero (African monkey kidney cell) cell line were cultured in liquid medium DMEM (Dulbeccos modified eagles medium) supplemented 10% Fetal Bovine Serum (FBS), 100g/ml penicillin and 100g/ml streptomycin, and maintained under an atmosphere of 5% CO2 at 37C.

2.4 MTT assay

The K1, K2, K3 and K4 samples were tested forin vitrocyto-toxicity, using A498 and Vero cells by 3-(4,5-dimethylth-iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [28]. The synthesized K1, K2, K3 and K4 samples were sus-pended with sterile phosphate buffer (PBS) and treated with various concentrations 10100g/ml in a serum free DMEM medium to treat the chosen cell line A498 and Vero cell. Each sample was replicated three times and the cells were incubated at 37C at 24h and each well, 20l of 5mg/ml MTT in phosphate buffer (PBS) was added. The

absorbance for each well was measured at 570nm using a micro plate reader (Thermo Fisher Scientific, USA) and the percent K1, K2, K3 and K4 samples cell viability and IC50 value was calculated using GraphPad Prism 6.0 soft-ware (USA). The data were collected for three replicates. The percentage of inhibition was calculated from this data using the formula

% of inhibition =mean OD of untreated cells (control) mean OD of treated cells

mean OD of untreated cells (control) 100.

nanopowder. The obtained nanopowder was annealed at 700C for 5h. The above procedure was followed for the preparation of the Ce, and Nd doped ZnO samples. Thus, ZnO (K1), La doped ZnO (K2), Ce doped ZnO (K3) and Nd doped ZnO (K4) samples were obtained. Figure1 shows the schematic diagram of synthesized K1, K2, K3 and K4 samples.

2.5 Characterization studies

The K1, K2, K3 and K4 samples were analysed by X-ray diffractometer (model: XPERT PRO PANalytical). The morphological features of the sample were measured by Field emission scanning electron microscopy (Model: Carl Zess 55) with EDAX (Ultra 55). The FT-IR spectrum was recorded in the range of 4004000cm1 by using Perkin-Elmer spectrometer. Ultravioletvisible spectra of the sample was measured on a Perkin-Elmer UV-Lambda 25 spectrophotometer (Perkin-Elmer, Norwalk, Connecticut). The PL emission study of the sample was carried out using Horiba JobinYVON spectrofluorometer (model: FLUORO-MAX-4, 450W high pressure Xenon lamp as the excitation source, photomultiplier at a range 325550nm). The XPS measurements were performed with an XPS instrument (Carl Zeiss) under-high vacuum with Al K excitation at 250W. To obtain information on defects and vacancies, EPR was recorded using X-band JEOL JES-RE1X at the room temperature.

3 Results anddiscussion

3.1 Xray diffraction studies

Figure2 shows the XRD patterns of the K1, K2, K3 and K4 samples using G. sylvestre leaves extract. The XRD peaks position are located at (100), (002), (101), (102), (110), (103), (112), (201), (004) and (202) for ZnO NPs, retained hexago-nal wurtzite structure of ZnO NPs with the p63mc space group corresponding to JCPDS data (Card No. 36-1451). In the case of REM doped ZnO NPs, there is no impurity phase observed in La3+ and Nd3+ samples. Furthermore, the ZnO doped with Ce NPs have one additional peak observed corresponding to 2 = 28.573 (JCPDS No 34-0394). This is due to the partial oxidation of Ce3+ into Ce4+, through the formation of CeO2. The XRD parameters like lattice con-stant, atomic peaks factor c/a, Cos(), position parameter (u) and bond length (L) estimated through the literature

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[29] and the values are given in the Table1. The small shift is observed for the REM doped ZnO NPs compared to the pure ZnO NPs.

The crystallite size (D) of pure ZnO NPs is determined by the X-ray line broadening method using the Scherrers equation,

(1)D =k

Dcos

where D is the size in nanometers, is the wavelength of radiation (1.5406 for CuK), k is a constant (0.94), D-is the peak width at half-maximum intensity and is the peak position.

The average crystalline size observed at 38, 33, 27 and 23nm correspond to K1, K2, K3 and K4 samples respec-tively. The REM doped ZnO NPs possess decreased size than the pure ZnO NPs, which may be due to alteration in the host ZnO matrix through the foreign impurities i.e., La3+, Ce3+, and Nd3+.

3.2 Morphology andelemental composition studies

Figure3ad shows the FESEM images for K1, K2, K3 and K4 samples. From the FESEM images, the K1, K2, K3 and K4 samples form a spherical, spindle, hexagonal and flake like nanostructures. The average particles size were observed at 138nm, 52nm, 59nm, and 63nm for K1, K2, K3 and K4 samples respectively. The average thicknesses was reduced for REM ions doped ZnO as compared to the pure ZnO NPs respectively. The reduction in thickness is attributed to the distortion in the ZnO matrix incorpo-rated with rare earth metal ions like La3+, Ce3+, and Nd3+. These doping materials are of different ionic radii such as La3+(1.061), Ce3+(1.034) and Nd3+(0.995) and hence, the substitution of the REM with ZnO matrix obviously changes the morphology of the REM doped ZnO nano-particles namely K1, K2, K3 and K4 samples [29].

The chemical composition of K1, K2, K3 and K4 samples are shown in Fig.3eh. In the case of doping samples K2, K3 and K4, the atomic percentage of La, Ce, and Nd are estimated as 11.90%, 7.18%, and 9.98% respectively. For K1 sample atomic percentage of Zn and O are observed at 83.90% and 16.10% respectively. For REM ions (La3+, Ce3+, and Nd3+)-doped ZnO NPs, zinc percentage increases whereas oxygen percentage decreases as compare to the pure ZnO NPs. The chemical composition values are given in the Table2.

30 60

(202

)

(004

)(201

)(112

)(103

)

(110

)

(102

)

2 Theta Degree

K1

(100

)(0

02)

(101

)

K2

Inte

nsity

(a.u

.)

K3

#

K4

Fig. 2 X-ray powder diffraction patterns of K1, K2, K3 and K4 sam-ples

Table 1 X-ray diffraction parameter values of the K1, K2, K3 and K4 samples

Samples Lattice parameter values (nm)

Atomic packing factor (c/a)

Volume (V) ()3 Cos Position parameter (u)

Bond length (ZnO) L ()

Average crystal-lite size D (nm)

a c

K1 0.3255 0.5215 1.6017 47.876 0.9459 0.3798 1.5569 38K2 0.3251 0.5209 1.6025 47.689 0.9578 0.3798 1.5555 33K3 0.3258 0.5217 1.6011 47.988 0.9465 0.3800 1.5579 27K4 0.3251 0.5214 1.6039 47.733 0.9464 0.3799 1.5551 23

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3.3 Fourier transform infrared (FTIR) spectroscopic studies

Figure4 shows FT-IR spectra of K1, K2, K3 and K4 samples. In the present investigation, wide OH stretching band has been observed at 3420cm1 for K1 sample, which may be surface absorbed water molecule [30]. The asym-metric CH stretching band is located at 2978cm1 for K1 sample. The narrow intense HOH bending centred

Fig. 3 Morphology of ad K1, K2, K3 and K4 samples and elemental composition of ehK1, K2, K3 and K4 samples

Table 2 The elemental composition of the synthesized K1, K2, K3 and K4 samples

Samples at%

Zn O Doping amount Total (%)

K1 83.90 16.10 100K2 76.92 11.17 11.90 (La) 100K3 76.29 16.52 7.18 (Ce) 100K4 71.05 18.97 9.98 (Nd) 100

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at 1571cm1. The medium intensity band 1424cm1 is attributed to C=O symmetric stretching. The OH asym-metric stretching bands found to be 1044 and 1014cm1 for K1 sample. The weak MetalOxygen (ZnO) vibra-tion frequencies are observed at 878 and 817cm1 for

K1 sample. The medium intense peak at 467cm1 was recognized as the ZnO stretching band. The REM doped ZnO NPs vibration frequencies small shift occur as com-pared to pure ZnO NPs. The metaloxygen (ZnO) vibra-tion frequency (467461 cm1) (467452 cm1) and (467439cm1) for K2, K3, and K4 samples respectively, due to the REM La3+, Ce3+ and Nd3+ ions substitution in the ZnO matrix.

3.4 UVVis spectroscopic studies

UVVisible spectra of K1, K2, K3 and K4 samples are shown in Fig.5. 3mg of pure and doped samples were uniformly dispersed in distilled water and the solution was ultra sonificated for 20min before recording UVVis absorbance spectra. The K1, K2, K3 and K4 samples exhib-ited strong absorption edge peaks at 378, 377, 376, and 374nm respectively. The REM doped ZnO NPs are blue shifted when compared to pure ZnO NPs, which may be due to the doping induced effects. The band gap energy are calculated using Tauc relation [31]. A plot (Fig. 6) between (h)2 and photon energy (eV) is drawn for K1, K2, K3 and K4 samples. The optical band gap of K1, K2, K3 and K4 samples are observed at 2.2, 1.8, 2.1 and 2.0eV respectively. The K1, K2, K3 and K4 samples band gap results are compared with commercially available TiO2 (for 3.1eV) and ZnO (for 3.37eV) [32]. The green synthe-sized K2 sample band gap energy (for 1.8eV) is lower than the commercially available TiO2 and ZnO NPs. This result showed that K2 sample can be more effectively used as photocatalyst.

3.5 Photoluminescence (PL) studies

Figure7 shows the photoluminescence spectra of K1, K2, K3 and K4 samples using an excitation wavelength of 325nm. In the case of K1 sample, the emission wave-lengths are observed at 421, 451, 465, 489 and 516nm respectively. The violet emission centered at 421nm is ascribed to an electron transition from a shallow donor level of the natural zinc interstitials to the top level of the valence band [33]. The two blue emissions located at 451 and 465nm are due to the singly ionized Zn vacan-cies [34]. The blue green emission observed at 489nm is ascribed to the transition between the oxygen vacancy and interstitial oxygen [35]. Finally green emission observed at 516nm, corresponds to the singly ionized oxygen [36, 37].

The PL emission band values for K1, K2, K3 and K4 NPs are given in Table3. Green emission band disappear for K4 sample as compared to K1 sample, due to the distortion

4000 3500 3000 2500 2000 1500 1000 500

Wavenumber (cm-1)

K1

K2

K3

Tra

nsm

ittan

ceK4

Fig. 4 FTIR spectrum of K1, K2, K3 and K4 samples

400 600 8000.3

0.4

0.5

0.6

0.7

0.8

Abs

orba

nce

(a.u

)

Wavelength (nm)

K1 K2 K3 K4

Fig. 5 Absorption spectra of K1, K2, K3 and K4 samples

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in the host ZnO lattice by the REM ion impurities. How-ever for the K2 sample, green emission values (522nm) increased as compared to the K1 (516nm) and K3 (510nm) samples respectively. These changes in emissions, confirm that REM (La3+, Ce3+ and Nd3+) ions incorporate with ZnO matrix.

3.6 XPS studies

XPS spectra of K1, K2, K3 and K4 samples are shown in Fig.8. The Zn (2p), O (1s), La (3d), Ce (3d) and Nd (3d) oxidation states were identified using XPS spectra. The Zn (2p) singlet split in two doublets, such as Zn 2p1/2 and Zn 2p3/2 observed at 1045.327 and 1022.214eV respec-tively, which is attributed to the ZnO matrix for the K1 sample had Zn2+ being bound to oxygen [29]. The K3 and K4 samples binding energy values are increased. In the case of K2 sample binding energy values are decreased,

due to the ions residing partially in the tetrahedral Zn positions [29].

Figure9 shows the O (1s) spectra of K1, K2, K3 and K4 samples. The green synthesized K1 sample of O (1s) signal observed at 531.115, 532.710 and 533.821eV respectively. The lower binding energy O (1s) sig-nal centre at 531.115 eV, which may be O2

ion in the wurtzite. The middle and higher binding energy located at 532.710 and 533.821eV are attributed to the loosely-bound oxygen, like absorbed O2 or adsorbed H2O on the ZnO surface. The REM doped ZnO NPs bind-ing energy of O (1s) values are observed as positional shift which is not observed in the case of K1 sample. This alteration may be charge due to the transfer effi-ciency from Zn2+ to O2

ions. These results strongly affected surface defects and vacancies, leading to increasing the charge-transferring efficiency in the metal ions.

Fig. 6 Tauc plots of (h)2 versus photon energy used to estimate optical band gap of K1, K2, K3 and K4 samples

1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5

0

2

4

6

8

10

(h)

2

Photon energy (eV)

K1

2.2eV

1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0

1

2

3

4

(h)

2

Photon energy (eV)

K2

1.8eV

1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5

0

2

4

6

8

10

(h)

2

Photon energy (eV)

K3

2.1eV

1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5

0

2

4

6

8

10

(h)2

Photon energy (eV)

K4

2.0 eV

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The 3d spectra for K2, K3 and K4 samples are shown in Fig.10ac. The La (3d) signal split into La 3d5/2 and La 3d3/2 observed at (836.42 and 839.92eV) and (853.34 and 856.35eV) for K2 sample respectively [38]. The Ce (3d) signal is divided into Ce 3d3/2 and Ce 3d5/2 state

located at 906.40 and 879.34eV for K3 sample respec-tively. The signals for Nd (3d) are found at 976.28, 982.35, 992.11 and 1003.01eV for K4 sample. It can be seen that La3+, Ce3+ and Nd3+ ion substitution in ZnO matrix, not only changes the atomic arrangement but also gradually tunes their electronic structures.

3.7 Antibacterial activity

The antibacterial activities of green synthesized ZnO (K1), La doped ZnO (K2), Ce doped ZnO (K3) and Nd doped ZnO (K4) NPs are treated with concentration 1.5mg/ml and it is shown in Fig.11. Day by day, more number of researchers are focused to study antibacterial activity of ZnO and doped ZnO NPs. But their doped concentration and their antibacterial results are varied. So, currently we

400 450 500 550 6000

2

4

6

8

10

PL In

tens

ity (a

.u.)

Wavelength (nm)

K1 420 nm 451 nm 466 nm 490 nm 522 nm Peak sum

400 450 500 550 6000

2

4

6

8

10

PL In

tens

ity (a

.u.)

Wavelength (nm)


400 450 500 550 6000

2

4

6

8

10

PL In

tens

ity(a

.u.)

Wavelength (nm)


350 400 450 500 5500

2

4

6

8

10

12

PL In

tens

ity (a

.u.)

Wavelength (nm)


Fig. 7 PL emissions spectra of K1, K2, K3 and K4 samples using the excitation wavelength at 325nm

Table 3 Gaussian decomposed photoluminescence emission val-ues of K1, K2, K3 and K4 samples

K1 (nm) K2 (nm) K3 (nm) K4 (nm)

421 420 420 366451 451 450 395465 466 464 408489 490 485 429516 522 510 466

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focused on the enhanced antibacterial activity of ZnO NPs. From this study, the K2 sample shows more antibac-terial effect than K1, K3 and K4 samples. Table4 shows a comparison between present and reported concentra-tion values of various metals doped ZnO NPs required to inhibit the growth of human pathogens [31, 3943]. The zone of inhibition (ZOI) of human pathogens is shown in Fig.12. The photo-generation of ZnO NPs reactive oxygen species (ROS) are accountable for various factors such as surface area, oxygen vacancies, and Zn2+ ions release [43]. The K1, K2, K3 and K4 NPs exhibits antibacterial activity as shown in Fig.12. However, the K2 sample shows the highest antibacterial activity. In early report, the appropri-ate crystallite size (ca. 33nm) caused higher antibacterial effects [29, 44]. From XRD results the particles size of the NPs are found to be 38, 33, 27 and 23nm for K1, K2, K3 and K4 samples respectively. The K2 sample exhibits 33nm for crystallite size, is ascribed to higher antibacterial activity. From the antibacterial activity, the NPs with uneven sur-faces and rough edges have been found to adhere to the

bacterial wall and cause damage to the cell membrane [29]. From the FESEM image, it clear shows different mor-phologies like spherical, spindle, hexagonal and flake structures for K1, K2, K3 and K4 samples respectively. La-doped ZnO NPs [K2] have uneven ridges at their outer sur-face which led to antibacterial activity, whereas the other NPs have smooth surfaces, which indicates that antibac-terial activity is effective in uneven ridged surfaces. The EPR spectra provides information about the native defects in K1, K2, K3 and K4 samples as shown in Fig.13. In early literatures, [28, 45, 46] the ZnO NPs (K1) higher intensity of the signal are associated with more oxygen vacancies (Vo) in it. Therefore, according to Fig.13, the higher intensity are observed for K1, K3 and K4 samples as compared to K2 samples. This result shows that the amount of oxygen vacancies in the K2 sample is more than that in the K1 sample. From antibacterial test, we conclude that K2 sam-ple render an effective antibacterial agent as compared to the K1, K3 and K4 samples. Based on the comparative statement, the present study confirmed that the pure and doped ZnO NPs exhibit moderate antibacterial activities respectively. It is worth to mention that all the samples exhibit strong antibacterial activity towards both G+ and G bacterial culture.

3.8 In vitro toxicity studies onnormal vero cell versuskidney cancer cell line

The ZnO NPs is a wide band-gap semiconductor and photo excitation under the UV lights, whose energy has greater than the band gap energy [47]. The photo excita-tion of ZnO initiates electron transfer from the valence band to the conduction band, its creating an elec-tronhole pair. In general, the holes in the valence band act as oxidants and thus generate hydroxyl radicals ( OH ) upon reaction with water. The electrons in the conduc-tion band reduce oxygen to produce superoxide anions ( O

2 ) [47]. Reactive oxygen species (ROS) generation by

ZnO NPs upon irradiation with UV light has been utilized for photo-triggered anticancer activities via ROS-induced damage the cell membranes, mitochondria, proteins, and DNA [4851].

The cytotoxicity of the K1, K2, K3 and K4 samples were tested at various concentration 10100g/ml for A498 (Kidney carcinoma cell) and Vero (African green monkey kidney cell) cell lines. The IC50 value of (41.74, 35.86, 40.05 and 63.08g/ml) and (55.27, 49.69, 56.83 and 51.10g/ml) (evaluated after 24h) of K1, K2, K3 and K4 samples against A498 and Vero cells was (p 0.05 p < 0.01). The La-doped ZnO (K2) sample showed a highly effective cytotoxic activity against A498 and Vero cells

1010 1020 1030 1040 1050 1060

Binding Energy (eV)

K1 1022.214 1045.327 Peak sum

Inte

nsity

(a.u

.)

K2 1022.072 1045.088 Peak sum

K3 1022.317 1045.365 Peak sum

K4 1022.358 1045.450 Peak sum

Fig. 8 XPS spectra of Zn (2p) for K1, K2, K3 and K4 samples

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(Fig.14a, b). Table5 shows a comparison between our and earlier reported IC50 values of ZnO NPs [42, 5258]. Cell morphological changes were observed for light microscope with different concentrations 10, 50 and 100g/ml (Fig.15).

As per the early discussion, the cytotoxicity effect of ZnO NPs potentially depends on the attendance of higher ROS, ZnO induced a reduced band gap due to

the increased redox capability. The enhancement in the anticancer activity in La doped ZnO NPs is due to the increased production of the reactive oxygen spe-cies (ROS) in the presence of La3+ ions and ZnO in the presence of UV light [59]. However, La doped ZnO (K2) sample, show enhanced ability to produce photo gen-erated holes (h+), resulting in stronger anticancer effect than ZnO (K1) sample Fig.16. Reactive oxygen species

Fig. 9 XPS spectra of O (1s) for K1, K2, K3 and K4 samples

525 530 535 540

Inte

nsity

(a.u

.)

Binding Energy (eV)

K1 (O1s)

531.115eV

532.710eV

533.821eV

Peak sum

(a)

525 530 535 540

Inte

nsity

(a.u

.)

Binding Energy (eV)

K2 (O 1s)

530.177eV

532.712eV

533.105eV

Peak sum

(b)

525 530 535 540

Inte

nsity

(a.u

.)

Binding Energy (eV)

K3 (O 1s)

530.624eV

532.833eV

533.028eV

Peak sum

(c)

525 530 535 540

Inte

nsity

(a.u

.)

Binding Energy (eV)

K4 (O 1s) 530.170eV 532.886eV 534.433eV Peak sum

(d)

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(ROS), such as hydrogen peroxide (H2O2), superoxide anion ( O

2 ), hydroxyl radicals ( OH ), and organic hydro

peroxides (OHPs) are toxic to the cells as they damage cellular constituents such as DNA, lipids, and proteins and also lanthanum ions interact with thiol groups in proteins, ensuing in inactivation of respiratory enzymes and leading to the production of more reactive oxygen species (ROS) Fig.17.

4 Conclusion

ZnO NPs (K1) and rare earth metal (REM) ions (La3+, Ce3+ and Nd3+) doped ZnO NPs were prepared through green method using Gymnema sylvestre leaves extract. Syn-thesized K1 (ZnO NPs), K2 (La doped ZnO NPs), K3 (Ce doped ZnO NPs) and K4 (Nd doped ZnO NPs) samples exhibit hexagonal wurtzite structure. Elemental oxidation state of Zn (2p), O (1s), La (3d), Ce (3d) and Nd (3d) were observed using XPS spectra. Morphological and elemental

Fig. 10 XPS spectra of 3d for K2, K3, and K4 samples

835 840 845 850 855 860

Inte

nsity

(a.u

.)

Binding Energy (eV)

La (3d) 836.42 eV 839.92 eV 853.34 eV 856.35 eV Peak sum

(a)

920 910 900 890 880

Inte

nsity

(a.u

.)

Binding Energy (eV)

Ce (3d) 909.54 eV 903.82 eV 888.85 eV Peak sum

(b)

980 990 1000 1010

Inte

nsity

(a.u

.)

Binding Energy (eV)

Nd (3d) 976.28 eV 982.35 eV 992.11 eV 1003.01 eV Peak sum

(c)

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composition were identified for the synthesized K1, K2, K3 and K4 samples using FESEM and EDAX spectra. Optical properties were estimated through UVVis and PL spec-tra. From the antibacterial activity, tested against clinical

pathogen using K1, K2, K3 and K4 samples. The K2 samples exhibits highest antibacterial effects as compared to other K1, K3 and K4 samples.

Fig. 11 The progress of antibacterial activity of K1, K2, K3 and K4 samples

Table 4 Comparative antibacterial values of present and various metals doped ZnO NPs on different bacterial strains

S.no Concentration Materials Size (nm) Bacteria

Present study1. 1.5mg/ml 0.002% of La in ZnO 33 Staphylococcus aureus

Streptococcus pneumoniaeEscherichia coliKlebsiella pneumoniaePseudomonas aeruginosa

2 0.002% of Ce in ZnO 273 0.002% of Nd in ZnO 23

Previous studies1 100 (l) 0.03% of La in ZnO [39] 11.46 Staphylococcus aureus2 1 (mg/ml) 0.01% of La in ZnO [40] 38 Staphylococcus aureus

Streptococcus pneumoniae3 10 (g/ml) 0.03% of Ce in ZnO [41] 11.56 Staphylococcus aureus4 1 (mg/ml) 0.05% Of Ce In ZnO [42] 32 Staphylococcus aureus

Streptococcus pneumoniae5 800 (g/ml) 0.003% of Nd in ZnO [43] 33 Escherichia coli, Klebsiella pneumoniae6 2 (mg/ml) 0.03% of Nd doped in ZnO [31] 35.2 Pseudomonas aeruginosa

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In vitro cytotoxicity effect were observed for A498 (human kidney carcinoma cell) and Vero (monkey kid-ney cell) lines. From practical therapeutic application, significant improvements are required to reduce the IC50 and to improve the cell specificity. The IC50 val-ues obtained are (41.74, 35.86, 40.05 and 63.08g/ml) and (55.27, 49.69, 56.83 and 51.10g/ml) for K1, K2, K3 and K4 samples against A498 and Vero cells line. Minimum toxicity percentage was observed for synthesized all K1, K2, K3 and K4 samples using Vero cells. To traverse new strategies to develop the next generation of drugs or agents to control bacterial infections and cytotoxic effects the antibacterial and anticancer properties of ZnO and REM-doped ZnO NPs were examined.

Fig. 12 The zone of inhibi-tion formed around each disc, loaded with test samples indi-cated the antibacterial activity of a S. aureus, b S. pneumoniae c E. coli d P. aeruginosa e P. vulgaris f K. pneumoniae and g S. sydenteriae for the K1, K2, K3 and K4 samples

1.6 1.8 2.0 2.2 2.4

-6.0x106

-4.0x106

-2.0x106

0.0

2.0x106

4.0x106

Inte

nsity

(a.u

.)

G factor

K1 (1.9498) K2 (1.9617) K3 (1.9589) K4 (1.9566)

Fig. 13 EPR spectra of K1, K2, K3 and K4 samples

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Fig. 14 a Effect of K1, K2, K3 and K4 samples on the cytotoxicity property in human A498 cell (Kidney carcinoma cell). b Effect of K1, K2, K3 and K4 samples on the cytotoxicity property in Vero cell (African green monkey kidney cell)

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Table 5 Comparative IC50 values of present and various metals doped ZnO NPs on cytotoxic response between the different cell types

Sample IC50 concentration Cell line References

ZnO and La, Ce and Nd doped ZnO NPs (Present study)

41.74, 35.86, 40.05 and 63.08g/ml Vero cells Present study55.27, 49.69, 56.83 and 51.10g/ml Kidney carcinoma cell

ZnO-2 and ZnO-4 41.85g/ml and 137.6g/ml HeLa cell lines [52]ZnO 280g/ml Human lung cancer cell line

(A549)[42]

Zn0.95 Ce0.05 O 82g/mlZn0.90 Ce0.10 O 68g/mlZn0.85 Ce0.15 O 76g/mlZnO 121g/ml MCF-7 Breast cancer cell line [53]ZnO 52.80g/ml Human myeloblasic leukemia

cell HL 60[54]

ZnO 100g/ml Human breast cancer MCF-7 [55]ZnO 42.60g/ml Human liver adenocarcinoma

cell HepG2[56]

Fe-doped ZnO 37.20g/mlAg-doped ZnO 45.10g/mlPd-doped ZnO 77.20g/mlCo-doped ZnO 56.50g/mlDoxorubicin 20.10g/mlZnO U87 HeLa HEK Human cerebral glioma

tumor U87[57]

ZnO micro-flower composed of nanorods

61.6g/ml 118g/ml 52.80g/ml

125g/ml 128g/ml 250g/ml Cervical cancer HeLaZnO micro-flower composed

of thin sheets31.26g/ml 126g/ml 125g/ml

ZnO microspheres composed of nanoparticles

30g/ml 61g/ml 62.5g/ml Normal HEK cells

Silver Zinc oxide (Ag:ZnO) 20g/ml (MCF-7) Human breast cancer (MCF-7) [58]550g/ml (A549) Human lung cancer (A549)

Silver-zinc oxide nanocom-posite (CD-Ag:ZnO NC)

50g/ml (MCF-7)70g/ml (A549)

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Fig. 15 Kidney carcinoma cell (A498) and vero cells treated with K1, K2, K3 and K4 samples at the respective different concentration

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Fig. 16 The mechanism of the generation of reactive oxygen species from ZnO (K1) and La doped ZnO NPs (K2) induced by UV light

Fig. 17 Possible mechanisms underlying the cytotoxic activities leading to the cell death as caused by La doped ZnO NPs (K2)

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Acknowledgements The authors are thankful to the members of Management committee and Principal of Jamal Mohamed College for providing necessary facilities.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict of interest.

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Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://doi.org/10.1080/14328917.2017.1324379https://doi.org/10.1080/14328917.2017.1324379Enhancement ofantibacterial andanticancer properties ofpure andREM doped ZnO nanoparticles synthesized using Gymnema sylvestre leaves extractAbstract1 Introduction2 Experimental methods2.1 Synthesis ofpure andREM doped ZnO NPs byusing G. sylvestre leaves extracts2.2 Antibacterial assay2.3 Cell culture2.4 MTT assay2.5 Characterization studies3 Results anddiscussion3.1 X-ray diffraction studies3.2 Morphology andelemental composition studies3.3 Fourier transform infra-red (FT-IR) spectroscopic studies3.4 UVVis spectroscopic studies3.5 Photoluminescence (PL) studies3.6 XPS studies3.7 Antibacterial activity3.8 In vitro toxicity studies onnormal vero cell versuskidney cancer cell line4 ConclusionAcknowledgements References

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